Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized pathogen of human diarrhea. This organism has been implicated in sporadic diarrhea in developing and industrialized countries, in the persistent diarrhea syndrome in AIDS patients and children in the developing world, in traveler's diarrhea, and in various diarrheal outbreaks. The PI discovered this pathotype of diarrheagenic E. coli and has been the leader in describing the pathogenesis and epidemiology of this organism, This is a competing continuation of our fundamental work on the pathogenesis of EAEC infection. Overall, our objectives are to advance knowledge of EAEC pathogenesis, to better define true EAEC pathogens, to refine diagnostic methods and to identify protective immunogen. The three aims of this proposal seek to extend the most important and promising aspects of the work funded under the current award. Aim 1: Characterization of EAEC adhesion-Aggregation is the defining characterization of EAEC. Our observations suggest that aggregative adherence (AA) is a prelude to biofilm formation, which occurs in vivo and which can be modeled in vitro. In this aim, we will further elucidate fundamental aspects of EAEC adherence. Aim 2: The Regulation of EAEC virulence- AggR is a highly prevalent and conserved activator of AAF expression. However, nothing else is known of the regulation of EAEC virulence. Beginning with AggR, we will expand our studies of EAEC gene regulation. Aim 3: Reconstructing EAEC-We will use in vitro organ culture and T84 cell models of EAEC infection to answer the question: what genes are necessary and sufficient to confer the effects that we observe? The work under this award will greatly advance the current state of knowledge of EAEC and will result in the identification of pathogenetic mechanisms, of diagnostic reagents and in vaccine candidates.